Human T-cell lymphotropic virus type I (HTLV-I) encodes a 40-kD nuclear transactivating phosphoprotein, Tax1. Through interaction with cellular transcription factors, Tax1 regulates the level of viral and cellular gene expression. In order to identify cellular transcription factors which Tax1 might regulate or directly interact with, regulatory proteins which bind specifically to the viral LTR and activate transcription have been investigated. The c-ets proto-oncogene family members, Ets1 and Ets2, are sequence-specific transcriptional activators of the HTLV-I long terminal repeat (LTR). The HTLV-I LTR contains two distinct Ets1- responsive regions, Ets1-responsive region 1 (ERR-1) and Ets1-responsive region 2 (ERR-2). Cotransfection of Ets1 with reporter plasmids containing ERR-1 or ERR-2 upstream of a basal promoter resulted in an increase in transcriptional activity. By gel mobility shift assay, the interaction of Ets1 with the downstream ERR-1 binding region was found to be more stable than its interaction with the upstream ERR-2 region. By DNase I footprint, gel mobility shift and methylation interference analyses, ERR-1 was found to contain two Ets1 binding sites, ERE-A and ERE-B. Binding of Ets1 to these sites appears to result in a specific and sequential protection of a 37 nucleotide sequence of the HTLV-I LTR from -154 to -118. In view of the high level expression of Ets1 in lymphoid cells, the c-ets proto-oncogenes encode transcription factors which could play an important role in both basal and Tax1-mediated HTLV-I transcription. The members of the c-myb proto-oncogene family encode sequence-specific transcriptional activators. In T-cells, expression of c-myb and the related b-myb gene is induced following mitogenic stimulation. Using a purified recombinant protein, it has been demonstrated that the HTLV-I LTR is transactivated by Myb and contains six specific binding sites for the transcription factor. These data suggest a role for members of the Myb family as a link between transcriptional activation of the HTLV-I LTR and T-cell activation events.